Cholesterol oxidase

ABSTRACT

A cholesterol oxidase having a particular amino acid sequence and having a high substrate affinity and a working pH in an acidic range is produced by Brevibacterium sterolicum.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of application Ser. No.798,660, filed Nov. 26, 1991, now abandoned, which is acontinuation-in-part application of application Ser. No. 683,539, filedApr. 10, 1991 (now abandoned).

BACKGROUND OF THE INVENTION

The present invention relates to a novel cholesterol oxidase having ahigh substrate affinity and the working pH in an acidic range.

Cholesterol oxidase is an enzyme which catalyzes the reaction ofoxidizing cholesterol (5-cholesten-3-α-ol) as substrate into4-cholesten-3-one and hydrogen peroxide. The enzyme is used in aclinical test for quantitative determination of cholesterol in blood,etc.

It is known that cholesterol oxidase has been produced by amicroorganism belonging to the genus Schizophyrum, Streptoverticillium,Brevibacterium or Streptomyces in an industrial scale. However, it hasbeen pointed out that a known cholesterol oxidase used for quantitativedetermination of cholesterol in blood has a low substrate affinity.Hence, ere is a problem in quantitative determination accuracy where asample solution having a low cholesterol content is analyzed and where adiluted sample solution is assayed. It has been further pointed out thatthe working pH range for the activity of known enzymes is relativelynarrow so that when pH of a sample is set in an acidic range, forexample at around 6, in order to avoid an influence by other componentssuch as bilirubin, the enzyme activity is lowered. In addition,productivity of the aforementioned cholesterol oxidase-producingmicroorganisms is extremely low and multiple procedures for purificationof enzymes are required to remove protein impurities.

Therefore, it has been desired to develop a cholesterol oxidase having ahigh substrate affinity and a wide working pH range as well as amicroorganism capable of producing cholesterol oxidase in highproductivity without contamination with protein impurities.

As a result of extensive studies in cholestrol oxidases, it has now beenfound that a novel cholesterol oxidase having a high substrate affinityand a wide working pH in an acidic range can be produced in good yield,by culturing a microorganism which is constructed by the steps of:isolating, from a strain belonging to the genus Brevibacterium, a DNAfragment bearing genetic information involved in the production of adifferent isoenzyme in the substrate affinity, working pH range andisoelectric point from known cholesterol oxidase, inserting the DNAfragment into a vector DNA to prepare a recombinant DNA containing theDNA fragment, introducing the recombinant DNA into a microorganismbelonging to the genus Escherichia. The present invention has thus beenaccomplished.

SUMMARY OF THE INVENTION

The present invention provides a novel cholesterol oxidase (hereafterreferred to as cholesterol oxidase II) which has nucleotide sequence asdefined in the Sequence Listing by SEQ ID: No. 1 and the followingphysicochemical properties, a process for production of cholesteroloxidase II, and a method for quantitative determination of cholesterolin a sample.

(a) Activity

It catalyzes the reaction of oxidizing cholesterol in the presence ofoxygen to form hydrogen peroxide and 4-cholesten-3-one.

(b) Isoelectric point: pH 4.7

(c) Substrate specificity:

It acts on cholesterol, α-sitosterol, stigmasterol, pregnenolone,dehydroisoandrosterone and estradiol but does not act on vitamin D₃,cholic acid, androsterone, cholesterol linoleate or lanosterol.

(d) Working pH range and stable pH range:

The working pH is in a range of 5.0 to 7.5. The enzyme is stable in a pHrange of 5.3 to 7.5, when heated at 50° C. for 60 minutes.

(e) Optimum temperature: about 50° C.

(f) Conditions for inactivation by pH and temperature:

The enzyme is inactivated at a pH of 10.0 or more or at a pH of 4.0 orless when heated at 50° C. for an hour. The enzyme is also inactivatedby about 83% when heated at a pH of 7.0 and a temperature of 60° C. foran hour.

(g) Inhibition and stabilization:

The enzyme is inhibited by p-chloromercury benzenesulfonate, silvernitrate and o-hydroxyquinoline. Further in the presence of bovine serumalbumin, resistance to heat and stability during storage are improved.

(h) Michaelis' constant to cholesterol (Km value):

    3.0×10.sup.-5 M

(i) Molecular weight:

about 43,000 (gel filtration)

about 60,000 (electrophoresis)

66,586 (DNA sequence)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows relative activities of cholesterol oxidases I and II atrespective pH values after treated at 37° C. for 5 minutes. In thefigure, ◯ denotes the activity of cholesterol oxidase II and denotes theactivity of cholesterol oxidase I.

FIG. 2 shows pH stability of the purified preparation of cholesteroloxidase II.

FIG. 3 shows the working temperature of the purified preparation ofcholesterol oxidase II.

FIG. 4 shows thermostability of the purified cholesterol oxidase II. Inthe figure, ◯ denotes the thermostability at 50° C. and denotes thethermostability at 60° C.

FIGS. 5A and 5B show substrate saturation curve of cholesterol oxidasesI and II on the purified cholesterol. That is, FIGS. 5A and FIG. 5Bdenote the results obtained by use of cholesterol oxidase II andcholesterol oxidase I, respectively.

FIG. 6 shows the cleavage map for restriction enzymes of recombinantplasmid pnH10.

FIG. 7 shows elution pattern in ion exchange chromatography ofcholesterol oxidase II using DEAECellulofine. In the figure, ◯ denotesprotein concentration (OD 280 nm, mg/ml) of the eluate and denotesenzyme activity (U/ml) of the eluate.

FIG. 8 shows elution pattern in gel filtration of cholesterol oxidase IIusing Superose prep12 HR16/50. In the figure, ◯ denotes proteinconcentration (OD 280 nm, mg/ml) of the eluate and denotes enzymeactivity (U/ml) of the eluate.

DETAILED DESCRIPTION OF THE INVENTION

Cholesterol oxidase II is an enzyme newly isolated from Brevibacteriumsterolicum ATCC 21387 described in Japanese Published Examined PatentApplication No. 1190/73. The strain has been known to be a cholesteroloxidase-producing strain. Cholesterol oxidase II is a novel enzymehaving properties different from those of a known cholesterol oxidase(hereafter referred to as cholesterol oxidase I) derived from ATCC 21387strain. Comparison between Cholesterol oxidases II and I are givenbelow.

(1) Isoelectric point:

Cholesterol oxidase II has an isoelectric point of pH 4.7, whereas anisoelectric point of cholesterol oxidase I is pH 8.9.

(2) Substrate specificity:

Substrate specificity of cholesterol oxidases I and II is shown in Table1.

                  TABLE 1                                                         ______________________________________                                                         Relative Activity (%)                                        Substrate          I       II                                                 ______________________________________                                        Cholesterol        100     100                                                Dehydroisoandrosterone                                                                           41.4    13.0                                               Pregnenolone       22.4    32.9                                               β-Sitosterol  19.7    82.0                                               Stigmasterol       10.0    64.4                                               Estradiol          0       3.2                                                Vitamin D.sub.3    0       0                                                  Cholic acid        0       0                                                  Androsterone       0       0                                                  Cholesterol linoleate                                                                            0       0                                                  Lanosterol         0       0                                                  ______________________________________                                    

(3) Working pH for activity:

The working pH of cholesterol oxidase II is in a range of 5.0 to 7.5,whereas that of cholesterol-oxidase I is in a range of 6.0 to 7.5.

(4) Michaelis' constant to cholesterol (Km value):

The constant of cholesterol oxidase II is 3.0×10⁻⁵ M, whereas theconstant of cholesterol oxidase I is 1.1×10⁻³ M.

(5) Influence of inhibitor:

Relative activity is shown in Table 2, in the presence of an inhibitorat a concentration of 1 mM, when in the absence of the inhibitor, theactivities of cholesterol oxidases I and II are regarded as 100.

                  TABLE 2                                                         ______________________________________                                                            Relative Activity                                                             (%)                                                       Inhibitor             II      I                                               ______________________________________                                        p-Chloromercury benzenesulfonate                                                                    87.0    80.0                                            AgNO.sub.3            1.0     0                                               o-Hydroxyguinoline    91.3    --                                              ______________________________________                                    

(6) Molecular weight:

From the analysis using TSK G3000SW manufactured by Toyo Soda Mfg. Co.,Ltd., it is presumed that the molecular weight is 43,000 based on itselution pattern (gel filtration). From the analysis using SDS-PAGE, itis presumed that the molecular weight is 60,000 based on its mobility.The DNA sequence determination indicates that the molecular weight is66,586. In the case of cholesterol oxidase I, the molecular weight ispresumed to be 33,000 by analysis using Sephadex gel filtration carriermanufactured by Pharmacia Fine Chemicals, Inc., and is presumed to be55,000 by analysis using SDS-PAGE.

Cholesterol oxidase II can be produced by culturing a microorganismhaving an ability to produce cholesterol oxidase II, and belonging tothe genus Brevibacterium sterolicum. However, since an accumulatedamount of cholesterol oxidase II is very small, it is difficult toisolate and purify the enzyme from a culture obtained by culturing astrain of Brevibacterium sterolicum in an ordinary way. By isolatingchromosomal DNA from a microorganism belonging to the genusBrevibacterium sterolicum, shotgun-cloning a gene coding for cholesteroloxidase II in an appropriate host-vector system, and introducing avector DNA containing the gene into a suitable Escherichia coli straincholesterol oxidase II-producing strain having a high expressionactivity can be constructed.

Hereafter construction of the cholesterol oxidase II-producing strain isdescribed.

Shotgun-cloning of cholesterol oxidase II encoding gene isolated from amicroorganism belonging to the species Brevibacterium sterolicum can berecognized by directly determining the expressed enzyme activity usingEscherichia coli as a host. The direct determination of the expressedenzyme activity can be made, for example, by using cells of atransformant as direct enzyme source, or by the method which compriseslyzing cells with lysozyme etc. and determining the enzyme activityusing the resulting permeable cell, or by the method which comprisesincorporating cholesterol in a medium and observing the clear haloformation associated with oxidation of cholesterol by the enzymeactivity leaked out of the cells in a trace amount.

Chromosomal DNA containing the gene coding for cholesterol oxidase II isisolated from a microorganism belonging to the species Brevibacteriumsterolicum in a conventional manner, for example, by the methoddescribed in Manual of Molecular Biology Experiment (R. F. Schleif, P.C. Wensink, translated by Masaya Kawakami and Tatsumi Yamasaki,published by Kodansha Scientific, 1983).

Then, the chromosomal DNA obtained above is digested with appropriaterestriction enzymes to prepare a DNA fragment containing cholesteroloxidase II encoding gene. The DNA fragment may be incorporated in aconventional manner into a vector DNA digested with appropriaterestriction enzymes, by use of DNA ligase to prepare the recombinant DNAcontaining the cholesterol oxidase II encoding gene. As the vector DNAused herein, any plasmid can be used so long as it is autonomouslyreplicable in Escherichia coli. Inter alia, pUC13, pPROK-C, etc. arepreferably used. As examples of the restriction enzyme, BamHI, Sau3AI,etc. can be used. The cleavage site of Sau3AI causes the extrusive endhaving the same structure as the cleavage site with BamHI so thatligation for recombination is possible. Thus, the DNA fragment obtainedby subjecting chromosomal DNA to restriction digestion with Sau3AI canbe ligated with the vector DNA digested with BamHI. As DNA ligase, T4DNA ligase derived from T4 phage-infected Escherichia coli can beadvantageously used.

Then, the recombinant DNA obtained by the method described above isintroduced into Escherichia coli in a conventional manner, for example,by the method described in Molecular Cloning (T. Maniatis, E. F.Fritsch, J. Sambrook, Cold Spring Harbour Publishing Co., 1982).Selection of a transformant carrying the recombinant DNA containingcholesterol oxidase II-encoding gene is made as follows. That is,transformants are cultured in an LB solid medium containing 0.1%cholesterol, 0.1% Trion X-100 and 0.0025% ampicillin. Colonies aroundwhich a clear halo formation is formed are selected from the growingcolonies. Then, the colonies are inoculated on a microtiter plate toassay the presence of cholesterol oxidase activity. An example of thethus obtained recombinant plasmids is pnH10. The transformant carryingthe resulting plasmid pnH10, namely, Escherichia coli nH10, has beendeposited under Budapest Treaty with the Fermentation Research Instituteof the Agency, Industrial Science Technology of Japan as Escherichiacoli nH10 (FERM BP-2850) since Apr. 5, 1990.

The thus obtained cholesterol oxidase II-producing strain is cultured ina nutrient medium, whereby marked quantities of cholesterol oxidase IIare accumulated in the culture.

As the media for culturing the cholesterol oxidase II-producing strain,any of synthetic media and natural media containing carbon sources,nitrogen sources, inorganic compounds, etc. can be used. As the carbonsource, there are used various carbohydrates such as lactic acid,glycerol and molasses. The preferred amount of the carbon sources usedis approximately 5 to 70 g/1. As the nitrogen source, there are usedammonium sulfate, ammonium phosphate, ammonium carbonate, ammoniumacetate, or nitrogen-containing organic compounds such as peptone, yeastextract, corn steep liquor, casein hydrolyzate and meat extract. Theamount of the nitrogen source is preferably about 5 to 20 g/1. As theinorganic compounds, there are used, for example, sodium chloride,potassium dihydrogenphosphate, dipotassium hydrogen phosphate, magnesiumsulfate, magnesium choloride, etc. The preferred amount of the inorganiccompounds used is approximately 0.05 to 5 g/1. Culturing is carried outunder aerobic conditions such as shaking culture and agitation submergedculture. For the culturing, it is preferred to maintain a temperature of30° to 37° C. In general, a period for the culturing is about 16 to 40hours.

For collecting cholesterol oxidase II from the culture after completionof the culturing, the cells are collected from the culture describedabove by means of centrifugation, etc. The obtained cells arehomogenized by sonication, trituration using glass beads, grindingtreatment with French press, etc. to extract the enzyme. The extract istreated in a conventional manner such as salting out with ammoniumsulfate, chromatography using ion exchange resin, gel filtration andchromatography using hydroxyappatite adsorption resin. to obtainpurified cholesterol oxidase II. Cholesterol oxidase II accumulatedoutside the cells may be treated in the same manner as described aboveexcept for omitting the operation for cell homogenization.

The physicochemical properties of the thus obtained cholesterol oxidaseII are described in detail below. With respect to Cholesterol oxidaseII, the purified enzyme preparation obtained in Example 2 was used.

I. Activity

It catalyzes the reaction of oxidizing cholesterol in the presence ofoxygen to form hydrogen peroxide and 4-cholesten-3-one.

(a) Ascertainment of the formation of hydrogen peroxide

Cholesterol is oxidized on cholesterol oxidase II in the presence ofoxygen and then peroxidase, phenol and 4-aminoantipyrine are added tothe enzyme system, whereby a quinonimine pigment is formed in thereaction system.

Reaction composition:

    ______________________________________                                        3 mM cholesterol, 1.0% Triton X-100                                                                      1.0 ml                                             aqueous solution                                                              50 mM sodium cholate aqueous solution                                                                    0.3 ml                                             0.5 M Na--K phosphate buffer (pH 6.0)                                                                    0.3 ml                                             42 mM phenol aqueous solution                                                                            0.5 ml                                             2.4 mM 4-aminoantipyrine aqueous solution                                                                0.5 ml                                             2 mg/ml peroxidase (manufactured by                                                                      0.2 ml                                             Toyobo Ltd., specific activity                                                115 units per 1 mg of protein)                                                cholesterol oxidase II aqueous solution                                                                  0.2 ml                                             (0.5 unit)                                                                    ______________________________________                                    

The reagents given in Reaction composition above were mixed with eachother. While stirring the mixture at 37° C., the amount of producedhydrogen peroxide was determined by colorimetric determination ofproduced quinonimine pigment. That is, when hydrogen peroxide producedin the reaction system was determined by the method as described inClinical Chemistry, 20, 470 (1974), it was recognized that 0.191 mole ofhydrogen peroxide was formed from 0.15 μmole of cholesterol.

(b) Ascertainment of oxygen consumption

In the presence of oxygen, cholesterol oxidase II acts on cholesteroland an amount of oxygen consumed is measured by Warburg's manometer.

Reaction composition:

    ______________________________________                                        3 mM cholesterol, 1.5% Triton X-100                                                                      1.0 ml                                             aqueous solution                                                              50 mM sodium cholate aqueous solution                                                                    0.3 ml                                             0.5 M Na--K phosphate buffer (pH 6.0)                                                                    0.3 ml                                             42 mM phenol aqueous solution                                                                            0.5 ml                                             2.4 mM 4-aminoantipyrine aqueous solution                                                                0.5 ml                                             2 mg/ml peroxidase (manufactured by                                                                      0.2 ml                                             Toyobo Ltd., specific activity                                                115 units per 1 mg/of protein)                                                cholesterol oxidase II aqueous solution                                                                  0.2 ml                                             (0.5 unit)                                                                    ______________________________________                                    

The reagents given in the reaction composition above were mixed witheach other. While stirring the mixture at 37° C., a difference ingaseous pressure between the inside and outside of a reactor caused byconsumption of oxygen was compensated for by shift of the manometer in acylinder connected with the reactor at one end and connected with theouter air at another end. In this case, an amount of the manometerliquid shifted was determined. An amount of the manometer liquid shiftedwas converted into an amount of ideal gas in the standard state by thefollowing equation (Umbreit Burris Stauffer, Manometric and BiochemicalTechniques, 5th edition, Chapter 5). ##EQU1## wherein symbols denote thefollowing. V: amount of ideal gas consumed in the standard state (unit:μl)

P: atmospheric pressure when measured (unit: mmHg)

Pw: vapor pressure at a reaction temperature when measured (unit: mmHg)

T: reaction temperature when measured (unit: K°)

ΔVg: amount of manometer liquid shifted (unit: μl)

In the standard state, 1 μl of gas corresponds to 0.0446 μmole.

The results of measurement indicates that when 1.5 μmole of cholesterolwas oxidized on cholesterol oxidase II, 1.43 μmole of oxygen wasconsumed.

(c) Ascertainment of the formation of 4-cholesten-3-one

In the presence of oxygen, cholesterol oxidase II acted on cholesterol,and the reaction product was identified to be 4-cholesten-3-one asfollows.

Procedures for identification:

1) Reaction solution

    ______________________________________                                        50 mM cholesterol ethanol solution                                                                    6.0 ml                                                50 mM sodium cholate aqueous solution                                                                 5.0 ml                                                Triton X-100            1.5 ml                                                0.5 M Na--K phosphate buffer (pH 7.5)                                                                 5.0 ml                                                cholesterol oxidase II aqueous solution                                                               13.0 ml                                               (19 units)                                                                    Water                   19.5 ml                                               ______________________________________                                    

2) Reaction procedures

The reaction solution described above was subjected to reaction at 37°C. while shaking. Thirty minutes, 3 hours and 16 hours after, 50 μl eachof the reaction solution was collected and mixed with 50 μl of hexane.Thereafter 5 μl of the resultant supernatant of the mixture wascollected and provided for the subsequent procedure (3) foridentification.

3) Identification

As the result of thin layer chromatography (hereafter referred to asTLC) shown below, the product in the reaction solution was identified tobe 4-cholesten-3-one.

The thin layer plate used was silica gel G-60F-254 (trademark,manufactured by E. Merck A. G.) and a developing solvent is the solventsystem [hexane:ethyl acetate=3:2 (volume ratio)] After development, theplate was observed in terms of fluorescent emission under exposure to UVrays (wavelength of 254 nm) followed by phosphorus molybdenate reaction.It was thus recognized that Rf value of the reaction product coincideswith that of the authentic compound. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Identification of the Reaction Product by TLC                                 Color Development                                                                              Sample       Rf value                                        ______________________________________                                        Exposure to UV ray                                                                             Authentic    0.808                                           (wavelength 254 nm)                                                                            4-cholesten-3-one                                                             Reaction product                                                                           0.808                                           Phosphorus molybdenate                                                                         Authentic    0.808                                           reagent          4-cholesten-3-one                                                             Reaction product                                                                           0.808                                           ______________________________________                                         (Note) phosphorus molybdenate reagent: Organic compounds containing           carbons react with the reagent to give a black spot.                     

(d) Based on the amount of hydrogen peroxide formed, the amount ofoxygen consumed and identification of the reaction product as describedin (a), (b) and (c), it is recognized that the enzyme of the presentinvention acts on specifically cholesterol, that cholesterol wasoxidized into hydrogen peroxide and 4-cholesten-3-one; that is, thisenzyme has a cholesterol oxidase activity. ##STR1## wherein R represents##STR2## II. Working pH range and stable pH range

The working pH for the activity is in a range of 5.0 to 7.5. The workingpH range was determined by assaying the activity at each pH (phosphatebuffer, Tris-HCl buffer, sodium acetate buffer) after the reaction at37° C. for 5 minutes. In this case, pH ranges were also determined withrespect to cholesterol oxidase I. The results are shown in FIG. 1. Asshown in FIG. 1, the working pH range of cholesterol oxidase II wasobviously different from that of cholesterol oxidase I. The stable pHrange is 5.3 to 7.5. The pH range is determined by treating the enzymeat 50° C. for 60 minutes at each pH (phosphate buffer, Tris-HCl buffer,sodium acetate buffer) and then assaying the residual activity. Theresults are shown in FIG. 2.

III. Method for determination of titer

The amount of enzyme which catalyzes the reaction of decomposing 1 μmoleof cholesterol at 37° C. for one minute in the presence of oxygen isdefined as one unit. The titer is determined as follows. While shaking,the enzyme acts on cholesterol and 4-aminoantipyrine and phenol arereacted with the formed hydrogen peroxide in the presence of peroxidaseto form quinonimine pigment. Absorption of the thus formed quinoniminepigment in the visible region is measured to determine the amount ofhydrogen peroxide generated. Thus, the titer of the enzyme isdetermined. Hereafter specific activity is defined as activity per 1 mgof protein (unit/mg). Furthermore, an amount of enzyme protein isdetermined by absorbance at 280 nm (1 mg/ml when absorbance is 1).

a) Principle

The enzyme activity is determined by reacting hydrogen peroxidegenerated by the enzyme with 4-aminoantipyrine and phenol in thepresence of peroxidase and quantitatively determining the formedquinonimine pigment. The reaction is shown by the following equations(1) and (2). ##STR3## wherein R represents ##STR4## (b) Procedure

In a test tube, there are charged 1.0 ml of 3 mM cholesterol solution(which is prepared by adding 6 ml of mM cholesterol ethanol solution to94 ml of 1.05% Triton X-100 solution with stirring, heating the mixturefor 10 minutes on a water bath, cooling the mixture in water, addingdistilled water to the mixture until the total volume is 100 ml; and isnot allowed to be used 30 minutes or longer after preparation), 0.3 mlof 50 mM sodium bilate solution, 0.3 ml of 0.5 mM potassium-sodiumphosphate buffer (pH 6.6) , 0.5 ml of 42 mM phenol, 0.5 ml of 2.4 mM4-aminoantipyrine and 0.2 ml of 115 units/ml horseradish-derivedperoxidase. The mixture is agitated. After maintaining at 37° C. for 3minutes, 0.2 ml of an enzyme solution is added to the mixture. Whileshaking, the reaction is carried out at 37° C. for 5 to 10 minutes.Then, absorbance is measured at 500 nm (OD 500 nm).

(c) Calculation of titer

One unit of cholesterol oxidase II corresponds to an enzyme activitywhich decomposes 1 μmole of cholesterol at 37° C. for 1 minute. On theother hand, it is reported that absorption coefficient of 1 mMquinonimine is 5.33 [Clinical Chemistry, 20, 470 (1974)]. Therefore,titer (A) per 1 ml of enzyme solution to be determined is calculatedaccording to equation (2), by defining OD 500 nm for 3 ml of thereaction solution as described in (1) below.

(1) A difference obtained by subtracting [(OD 500 nm of reagentblank)--(OD 500 nm of the solution from which substrate has been removedfrom the reagent blank)] from [(OD 500 nm of the reaction solutioncontaining both enzyme and substrate)--(OD 500 nm of the reactionsolution from which substrate has been removed from the reactionsolution)] is defined to be ΔE.

(2) Titer of enzyme solution A (unit/ml) =ΔE 5.33 time (min)×3 ×dilutionrate

Note 1: Reagent blank means a solution obtained by removing enzymesolution from the reaction solution.

IV. Optimum temperature

The optimum temperature at a pH of 6.6 for 3 minutes of the reactiontime was examined. The results are shown in FIG. 3. The optimumtemperature is at about 50° C.

V. Inactivation by pH and temperature.

Cholesterol oxidase II is inactivated at pH of 8.5 or more. After heattreatment at pH of 7.0 for 60 minutes in 0.05M phosphate buffer, theresidual activity was determined. As shown in FIG. 4, the results revealthat the enzyme is inactivated by about 10% at 50° C. and by about 83%at 60° C.

VI. Substrate affinity

Michaelis' constant (Km value) of cholesterol oxidase II to cholesterolsubstrate was 3.0×10⁻⁵ M, whereas that. of cholesterol oxidase I was1.1×10⁻³ M. Reaction rates of the two enzymes were measured in variousconcentrations of cholesterol. The results are shown in FIG. 5. Km valueof cholesterol oxidase II was about the one-hundredth of that ofcholesterol oxidase I. It is indicated that affinity of cholesteroloxidase II to cholesterol is extremely high and more suited forquantitative determination of cholesterol. In addition, as isillustrated in FIG. 5, the saturation curve obtained by use ofcholesterol oxidase II is a type of so-called Michaelis-Menten. Thesubstrate activation phenomenon was not observed, while it was observedwith cholesterol oxidase I. It is concluded that cholesterol oxidase IIhas superior reactivity to cholesterol oxidase I in quantitativedetermination of serum cholesterol at a low concentration.

Michaelis' constant was determined from the Line weaver-Burk plot [referto J. Am. Chem, Soc., 56, 658 (1934)].

VII. Determination of the amino acid sequence and nucleotide sequence ofcholesterol oxidase II

The nucleotide sequence of cholesterol oxidase II is determined by themethod of Sanger [refer to F. Sanger, Science, 214, 1205 (1981)] usingSequenase Ver 2.0 [trademark, manufactured by United StatesBiochemical].

Hereafter the present invention is described in more detail, withreference to the examples.

EXAMPLE 1 Cloning of cholesterol oxidase II-encoding gene

1) Preparation of chromosomal DNA containing cholesterol oxidaseII-encoding gene

Brevibacterium sterolicum ATCC 21387 strain was cultured with shaking at30° C. for 3 days in 30 ml of an LB medium [10 g/l Bactotrypton, 8 g/lBacto-yeast extract (both were manufactured by Difco Co.), 5 g/l NaCl(pH 7.2)]. The resulting cells were collected by centrifugation at 4° C.for 10 minutes at 10,000 rpm using a cooling centrifuging machine(RPR20-2 rotor) manufactured by Hitachi Ltd. After washing the cellswith 10.3% sucrose solution, the solution was again centrifuged tocollect the cells. The whole chromosomal DNA was extracted by the methoddescribed in Current Topics in Microbiology and Immunology, 96 (1982) togive about 1 mg of chromosomal DNA.

2) Incorporation of chromosomal DNA fragment into vector DNA

72 μg of the whole chromosomal DNA obtained in 1) above was taken anddissolved in 1,000 μl of M buffer [10 mM Tris-HCl buffer (pH 7.5), 10 mMMgCl₂, 50 mM NaCl, 1 mM DTT (dithiothreitol)]. 3.6 units of restrictionendonuclease Sau3AI (manufactured by Takara Shuzo Co., Ltd.) was addedto the solution to effect restriction digestion at 37° C. for 30minutes. Then, the fractionation was carried out by 10 to 40% sucrosedensity gradient centrifugation according to the method described inMolecular Cloning supra. Centrifugation was performed at 20° C. for 16hours at 26,000 rpm using an ultracentrifuging machine (SRP 28 rotor)manufactured by Hitachi Ltd. The fractionation was carried out again andan part of each fraction was subjected to agarose gel electrophoresisaccording to the method described in Molecular Cloning supra todetermine the size of DNA fragments. The fractions containing the DNAfragments of 3 to 6 kb were collected followed by ethanol precipitation.Thereafter, the precipitates were dissolved in 60 μl of a ligationbuffer [66 mM Tris-HCl buffer (PH 7.6), 5 mM MgCl₂, 5 mM DTT, 1 mM ATP]to give the chromosomal DNA solution containing about 5 μg of the DNAfragment. Furthermore, 10 μg of pPROK-C (manufactured by Clone Tech Co.,Ltd.) used as a vector was dissolved in 300 μl of M buffer and 40 unitsof restriction endonuclease BamHI (manufactured by Takara Shuzo Co.,Ltd.) was added to the solution. The reaction was carried out at 37° C.for 3 hours to fully digest the vector. Then, 20 μl of 1M Tris-HClbuffer (pH 8.6) and 4 units of calf small intestine-derived alkalinephosphatase were added. The reaction was carried out at 37° C. for anhour for dephosphorylation. Further, by heating at 65° C. for 10minutes, the enzyme was inactivated. After ethanol precipitation, theprecipitates were dissolved in 80 μl of ligation buffer solution.Subsequently, 32 μl of a solution of chromosomal DNA digested withSau3AI was mixed with 5 μl of a solution of pPROK-C digested with BamHI.The ligation buffer was added until the whole volume was 75 μl, and 2units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) wasadded to the mixture. Ligation was performed at 16° C. for 16 hours togive various recombinant DNA mixtures.

3) Transformation of Escherichia coli

With the recombinant DNA mixtures obtained in 2) above, Escherichia coliwas transformed. The DNA-sensitive strain used for transformation wasprepared by the method of Hanahan et al. described in Journal ofMolecular Biology, 166, 577. Escherichia coli MM294 strain was culturedin LB medium overnight and 0.2 ml of the obtained culture was inoculatedon 20 ml of SOB medium [20 g/l Bactotrypton, 0.50 g/l Bacto-yeastextract (both were manufactured by Difco Co.), 10 mM NaCl, 2.5 mM KCl,10 mM MgCl₂, 10 mM MgSO₄ (pH 7.0)]. After culturing for 3 hours, theculture was transferred to a test tube of Falcon 2070 and settled in icefor 15 minutes. By centrifugation at 4° C., the cells were collected.The cells were suspended in 7 ml of TFB [10 mM Good MES buffer (pH6.20), 100 mM RbCl, 45 mM MnCl₂, 10 mM CaCl₂, 3 mM hexamine cobaltchloride] and the suspension was settled in ice for 15 minutes. Thecells were collected by centrifugation and again suspended in 1.6 ml ofTFB. Then, 56 μl of dimethylsulfoxide was added to the resultingsuspension. After gently stirring in ice for 5 minutes, 56 μl ofβ-mercaptoethanol was added and the mixture was gently stirred in icefor 10 minutes. Again 56 μl of dimethylsulfoxide was added and themixture was gently stirred in ice for 5 minutes. The thus obtained cellswere used for transformation as the DNA-sensitive strain. After 210 μlof the resulting solution containing DNA-sensitive strains was chargedin Falcon 2059 test tube, 10 μl of the recombinant DNA mixtures preparedin 2) was added thereto. After gently stirring, the mixture was settledin ice for 30 minutes. The mixture was heated for 90 seconds in athermostat of 42° C., then transferred into ice, and kept for 2 minutes.To the mixture was added 800 μl of SOC medium (SOB medium supplementedwith 20 mM glucose). The resulting culture was inoculated on LB solidmedium containing 0.1% cholesterol, 0.1% Triton X-100 and 0.0025%ampicillin followed by culturing at 37° C. overnight. The colonies grownwere obtained as transformants.

4) Selection of transformants carrying the recombinant DNA containingcholesterol oxidase II encoding gene

The recombinant DNA containing cholesterol oxidase II-encoding gene wasisolated from the transformants obtained in 3) described above by thefollowing method. The colonies which grew on the LB solid medium andaround which a clear halo formation was formed were selected. TheColonies were inoculated on a microtiter plate to assay the presence orabsence of cholesterol oxidase activity. The transformants having thecholesterol oxidase activity were inoculated on LB medium containing0.005% of ampicillin followed by culturing at 30° C. overnight. Aftercompletion of the culturing, the cells were collected. The obtainedcells were suspended in 10 ml of 0.05M phosphate buffer (pH 7.0) andthen disrupted for 10 minutes by ultrasonic cell disrupter (CellDisrupter, Model 200, manufactured by Branson Co., output: 40%). Aftercentrifugation, cholesterol oxidase activity of the resultingsupernatant was determined by the method described above. Thus,Escherichia coli nH10 having a high cholesterol oxidase activity wasobtained. From the transformant nH10, a recombinant plasmid DNA carriedby the transformant was recoverd to give pnH10. pnH 10 was cleaved withHindIII, PstI, SalI, XhoI, KpnI, ApaI, BamHI, MluI, EcoRI, SmaI and ScaIand the structure of pnH10 was recognized by agarose gelelectrophoresis. pnH10 had such a structure that DNA fragment of about3.0 kb had been incorporated into pPROK-C (cf. FIG. 6).

5) Analysis of cloned cholesterol oxidase II-encoding gene

It was ascertained by the following method that pnH10 was a recombinantplasmid containing a gene coding for isoenzyme different from knowncholesterol oxidase I produced by Brevibacterium sterolicum ATCC 21387.That is, recombinant plasmid pnH10 was prepared from nH10 strainaccording to the method described in Molecular Cloning (T. Maniatis, E.F. Fritsch, J. Sambrook, Cold Spring Harbour Publishing Co., 1982).After agarose gel electrophoresis, the gel containing pnH10 was dippedin a solution containing 1.5M NaCl and 0.5M NaOH at room temperature for30 minutes to denature double stranded DNA. By closely contacting thegel with a nylon filter, a solution containing 1.5M NaCl and 0.25M NaOHwas permeated through the back surface of the filter, whereby thedenatured DNA was transferred and fixed on the filter. The thus preparedfilter was reacted at 45° C. for 16 hours with hybridization buffer(53.0 g/l NaCl, 26.5 g/l sodium succinate, 10 mM EDTA, 5-fold Denhardtsolution, 0.5% SDS, 0.1 mg/ml thermally denatured bovine thymus-derivedDNA, pH 7.0) containing DNA probe obtained by labeling synthetic singleDNA having DNA nucleotide sequence deduced from the partial amino acidsequence of cholesterol oxidase I produced by Brevibacterium sterolicumATCC 21387, namely, Probe No. 1 as defined in the Sequence Listing bySEQ ID No. 2 and illustrated below: ##STR5## (wherein the 6th base isany one of T and C; the 9th base is any one of T and C; the 12th base isany one of A, G and T; the 15th base is any one of A and G; whichbecomes a mixture of 24 synthetic DNAs in combination); or Probe No. 2as defined in the Sequence Listing by SEQ ID No. 3 and illustratedbelow: ##STR6## (wherein the 3rd base is any one of A and G; the 9thbase is any one of A and G; the 12th base is any one of C and T; the15th base is any one of T, A and G; which becomes a mixture of 24synthetic DNAs in combination); with γ-³² P-ATP. Then, the solutioncontaining the probe was discarded and the reaction mixture wasthoroughly washed with 3-fold SSC at 65° C. The filter was closelycontacted with X ray film, which was exposed at -70° C. for one day. Byso-called autoradiography, the reactivity with pnH10 was examined. ProbeNo. 1 corresponds to the amino acid partial sequence of the polypeptidechain at the center of cholesterol oxidase I and probe No. 2 correspondsto the amino acid sequence at the C-terminal. The analyses ofautoradiography indicate that pnH10 did not react with Probe Nos. 1 andNo. 2, at all. The results reveal that the cholesterol oxidaseII-encoding gene contained in plasmid pnH10 is different from thecholesterol oxidase I-encoding gene.

EXAMPLE 2

Escherichia coli nH10 (FERM BP-2850) was inoculated on 3 ml of a seedmedium (pH 7.2 prior to sterilization) obtained by supplementing 0.005%of ampicillin to LB medium and cultured with shaking at 30° C. for 18hours. Three milliliters of the seed culture was put into each of five 2l-Erlenmeyer's flasks provided with baffles and containing 500 ml of themedium having the same components as the seed medium described above.Shaking culture was carried out at 30° C. for 16 hours. After culturing,the obtained culture was centrifuged at 10,000×g for 20 minutes with acooling centrifuging machine to give about 18.6 g (wet cell weight) ofthe cells. To the obtained cells were added 186 ml of 0.01M phosphatebuffer (pH 7.0, hereafter referred to as Buffer) to suspend the cells.Ultrasonication was performed for 30 minutes using an ultrasonicationhomogenizer (cell disrupter, Model 200, manufactured by Branson Co.;output of 75%, pulse retention time of 70%, mounted with a flat chip).After sonication, the disrupted mixture was centrifuged at 20,000×g for15 minutes with a cooling centrifuging machine and the supernatant wasobtained as the cell-free extract. Solid ammonium sulfate was added to194 ml of the obtained cell-free extract to 60% saturation. Whilekeeping pH at 7 with 1N NaOH solution, the mixture was gently stirred.After ammonium sulfate was completely dissolved, stirring was continuedfor further 30 minutes. Then, the solution was centrifuged at 20,000×gfor 15 minutes with a cooling centrifuging machine to give thesupernatant. The obtained supernatant was dialyzed to a cellophane tubeas a dialysis membrane for 12 hours per liter of Buffer. ThereafterBuffer was exchanged and dialysis was performed for further 12 hours toeffect desalting. After the dialysis, 98.5 ml of the crude enzymesolution was passed through a column (φ7.5×15 cm) of DEAE-Cellulofine(manufactured by Biochemical Industry Co., Ltd.), which had beenpreviously equilibrated with Buffer. By washing with 3 liters of Bufferat a flow rate of 480 ml/hr, unadsorbed protein was washed out. Next,NaCl concentration was changed linearly from 0 to 0.5M to elute theenzyme with density gradient. Among the eluted fractions, the fractionhaving a specific activity of 10 units/mg or more was collected. Elutionpattern is shown in FIG. 7.

The obtained active fraction was concentrated by adding ammonium sulfateto 60% saturation. The formed precipitates were dissolved in Buffer andthe solution was dialyzed as described above to effect desalting. Thesolution was further concentrated to about 2 ml by ultrafiltration. Theconcentrate was passed through a column (φ1.3×50 cm) of Superose prep12HRl 16/50 (manufactured by Pharmacia Fine Chemicals Co., Ltd.), Whichhad been previously equilibrated with 0.05M phosphate buffer (pH 7.0)supplemented with 0.3M NaCl and connected with 880PU pump (manufacturedby Nippon Bunko) to perform elution at a flow rate of 30 ml/hr. Amongthe eluted fractions, the fractions having a specific activity of 15units/mg or more were collected and dialyzed against 1 liter of Buffer.Elution pattern is shown in FIG. 8. The obtained active fraction waspassed through a column (φ3.2×4.0 cm) of hydroxyappatite (manufacturedby Biochemical Industry Co., Ltd.), which had been previouslyequilibrated with Buffer. By washing in Buffer at a flow rate of 100ml/hr, the activity was all recovered in the washing liquid. The washingliquid was concentrated by ultrafiltration and the concentrate was madethe purified enzyme preparation. The foregoing steps for purificationare summarized in Table 4.

                  TABLE 4                                                         ______________________________________                                                       Total    Total   Specific                                                     Activity Protein Activity                                                                             Yield                                  Step           (U)      (mg)    (U/mg) (%)                                    ______________________________________                                        Cell-free extract                                                                            1462     6940    0.21   100                                    Fraction with ammonium                                                                       2368     5990    0.40   162                                    sulfate (0-60%)                                                               DEAE-Cellulofine                                                                             844      57      14.8   57.7                                   Superose prepl2                                                                              740      39      19.1   51.0                                   Hydroxyappatite                                                                              404      20      20.4   28.0                                   ______________________________________                                    

Further in order to assay the purity of the purified enzyme preparation,the sample in the non-denatured state as obtained above was applied topolyacrylamide gel electrophoresis. The assay was carried out accordingto the method of Davis et al. [B. J. Davis et al., Annals of New YorkAcademy of Science, 121, 404 (1964)]. The results reveal that thepurified enzyme preparation was homogeneous.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES:3                                                   (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:1845 base pairs                                                    (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:double                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Genomic DNA                                                (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Brevibacterium sterolicum                                         (B) STRAIN:ATCC21387                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGACGCTCAACGACGAGCAGTTACGGCTGTCCCGGCGAGGATTCCTC48                            MetThrLeuAsnAspGluGlnLeuArgLeuSerArgArgGlyPheLeu                              15 1015                                                                       ACCGCGGGCGCTGCGGGCGCCGGCGTGCTGGCAGCCGGCGCACTCGGC96                            ThrAlaGlyAlaAlaGlyAlaGlyValLeuAlaAlaGlyAlaLeuGly                              20 2530                                                                       GGCTGGACCCCGGCCTTCGCCGTCCCTGCCGGTTCCGCCGGCTCCCTC144                           GlyTrpThrProAlaPheAlaValProAlaGlySerAlaGlySerLeu                              354 045                                                                       GGATCGCTCGGATCGACCGGGCCGGTCGCGCCGCTTCCGACGCCGCCG192                           GlySerLeuGlySerThrGlyProValAlaProLeuProThrProPro                              5055 60                                                                       AACTTCCCGAACGACATCGCGCTGTTCCAGCAGGCGTACCAGAACTGG240                           AsnPheProAsnAspIleAlaLeuPheGlnGlnAlaTyrGlnAsnTrp                              657075 80                                                                     TCCAAGGAGATCATGCTGGACGCCACTTGGGTCTGCTCGCCCAAGACG288                           SerLysGluIleMetLeuAspAlaThrTrpValCysSerProLysThr                              8590 95                                                                       CCGCAGGATGTCGTTCGCCTTGCCAACTGGGCGCACGAGCACGACTAC336                           ProGlnAspValValArgLeuAlaAsnTrpAlaHisGluHisAspTyr                              100105 110                                                                    AAGATCCGCCCGCGCGGCGCGATGCACGGCTGGACCCCGCTCACCGTG384                           LysIleArgProArgGlyAlaMetHisGlyTrpThrProLeuThrVal                              115120 125                                                                    GAGAAGGGGGCCAACGTCGAGAAGGTGATCCTCGCCGACACGATGACG432                           GluLysGlyAlaAsnValGluLysValIleLeuAlaAspThrMetThr                              130135140                                                                     CA TCTGAACGGCATCACGGTGAACACGGGCGGCCCCGTGGCTACCGTC480                          HisLeuAsnGlyIleThrValAsnThrGlyGlyProValAlaThrVal                              145150155160                                                                  ACGGCCGGTGCCGGCGCCAGCATCGAGGCGATCGTCACCGAACTGCAG528                           ThrAlaGlyAlaGlyAlaSerIleGluAlaIleValThrGluLeuGln                              16517017 5                                                                    AAGCACGACCTCGGCTGGGCCAACCTGCCCGCTCCGGGTGTGCTGTCG576                           LysHisAspLeuGlyTrpAlaAsnLeuProAlaProGlyValLeuSer                              180185190                                                                      ATCGGTGGCGCCCTTGCGGTCAACGCGCACGGTGCGGCGCTGCCGGCC624                          IleGlyGlyAlaLeuAlaValAsnAlaHisGlyAlaAlaLeuProAla                              195200205                                                                     GTCGG CCAGACCACGCTGCCCGGTCACACCTACGGTTCGCTGAGCAAC672                          ValGlyGlnThrThrLeuProGlyHisThrTyrGlySerLeuSerAsn                              210215220                                                                     CTGGTCACCGAGC TGACCGCGGTCGTCTGGAACGGCAACACCTACGCA720                          LeuValThrGluLeuThrAlaValValTrpAsnGlyAsnThrTyrAla                              225230235240                                                                  CTCGAGACG TACCAGCGCAACGATCCTCGGATCACCCCACTGCTCACC768                          LeuGluThrTyrGlnArgAsnAspProArgIleThrProLeuLeuThr                              245250255                                                                     AACCTCGGG CGCTGCTTCCTGACCTCGGTGACGATGCAGGCCGGCCCC816                          AsnLeuGlyArgCysPheLeuThrSerValThrMetGlnAlaGlyPro                              260265270                                                                     AACTTCCGTCA GCGGTGCCAGAGCTACACCGACATCCCGTGGCGGGAA864                          AsnPheArgGlnArgCysGlnSerTyrThrAspIleProTrpArgGlu                              275280285                                                                     CTGTTCGCGCCGAAGG GCGCCGACGGCCGCACGTTCGAGAAGTTCGTC912                          LeuPheAlaProLysGlyAlaAspGlyArgThrPheGluLysPheVal                              290295300                                                                     GCGGAATCGGGCGGCGCCGAGGCG ATCTGGTACCCGTTCACCGAGAAG960                          AlaGluSerGlyGlyAlaGluAlaIleTrpTyrProPheThrGluLys                              305310315320                                                                  CCGTGGATGAAGGTGTGGACG GTCTCGCCGACCAAGCCGGACTCGTCG1008                         ProTrpMetLysValTrpThrValSerProThrLysProAspSerSer                              325330335                                                                     AACGAGGTCGGAAGCCTCGG CTCGGCGGGCTCCCTCGTCGGCAAGCCT1056                         AsnGluValGlySerLeuGlySerAlaGlySerLeuValGlyLysPro                              340345350                                                                     CCGCAGGCGCGTGAGGTCTCCG GCCCGTACAACTACATCTTCTCCGAC1104                         ProGlnAlaArgGluValSerGlyProTyrAsnTyrIlePheSerAsp                              355360365                                                                     AACCTGCCGGAGCCCATCACCGACATG ATCGGCGCCATCAACGCCGGA1152                         AsnLeuProGluProIleThrAspMetIleGlyAlaIleAsnAlaGly                              370375380                                                                     AACCCCGGAATCGCACCGCTGTTCGGCCCGGCGATG TACGAGATCACC1200                         AsnProGlyIleAlaProLeuPheGlyProAlaMetTyrGluIleThr                              385390395400                                                                  AAGCTCGGGCTGGCCGCGACGAATGCCAACGA CATCTGGGGCTGGTCG1248                         LysLeuGlyLeuAlaAlaThrAsnAlaAsnAspIleTrpGlyTrpSer                              405410415                                                                     AAGGACGTCCAGTTCTACATCAAGGCCACGA CGTTGCGACTCACCGAG1296                         LysAspValGlnPheTyrIleLysAlaThrThrLeuArgLeuThrGlu                              420425430                                                                     GGCGGCGGCGCCGTCGTCACGAGCCGCGCCAAC ATCGCGACCGTGATC1344                         GlyGlyGlyAlaValValThrSerArgAlaAsnIleAlaThrValIle                              435440445                                                                     AACGACTTCACCGAGTGGTTCCACGAGCGCATCGAGTTC TACCGCGCG1392                         AsnAspPheThrGluTrpPheHisGluArgIleGluPheTyrArgAla                              450455460                                                                     AAGGGCGAGTTCCCGCTCAACGGTCCGGTCGAGATCCGCTGCTGCGG G1440                         LysGlyGluPheProLeuAsnGlyProValGluIleArgCysCysGly                              465470475480                                                                  CTCGATCAGGCAGCCGACGTCAAGGTGCCGTCGGTGGGCCCGC CGACC1488                         LeuAspGlnAlaAlaAspValLysValProSerValGlyProProThr                              485490495                                                                     ATCTCGGCGACCCGTCCGCGTCCGGATCATCCGGACTGGGAC GTCGCG1536                         IleSerAlaThrArgProArgProAspHisProAspTrpAspValAla                              500505510                                                                     ATCTGGCTGAACGTTCTCGGTGTTCCGGGCACCCCCGGCATGTTC GAG1584                         IleTrpLeuAsnValLeuGlyValProGlyThrProGlyMetPheGlu                              515520525                                                                     TTCTACCGCGAGATGGAGCAGTGGATGCGGAGCCACTACAACAACGAC 1632                         PheTyrArgGluMetGluGlnTrpMetArgSerHisTyrAsnAsnAsp                              530535540                                                                     GACGCCACCTTCCGGCCCGAGTGGTCGAAGGGGTGGGCGTTCGGTCCC1680                          Asp AlaThrPheArgProGluTrpSerLysGlyTrpAlaPheGlyPro                             545550555560                                                                  GACCCGTACACCGACAACGACATCGTCACGAACAAGATGCGCGCCACC1728                          AspProTyrThrAspAsnAspIleValThrAsnLysMetArgAlaThr                              565570575                                                                     TACATCGAAGGTGTCCCGACGACCGAGAACTGGGACACCGCGCGCGCT177 6                         TyrIleGluGlyValProThrThrGluAsnTrpAspThrAlaArgAla                              580585590                                                                     CGGTACCAACCAGGATTCGACCGGCATCGCGTGTTCACCAACGGATTC1824                           ArgTyrGlnProGlyPheAspArgHisArgValPheThrAsnGlyPhe                             595600605                                                                     ATGGACAAGCTGCTTCCGTAG1845                                                     MetAsp LysLeuLeuPro                                                           610615                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:17 base pairs                                                      (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Other nucleic acid Synthetic DNA                           (iii) HYPOTHETICAL:YES                                                        (iv) ANTI-SENSE:YES                                                           (x) PUBLICATION INFORMATION:                                                   (A) AUTHORS:Fujishiro, Kinya                                                 Ohta, Toshio                                                                  Hasegawa, Mamoru                                                              Yamaguchi, Kazuo                                                              Mizukami, Toru                                                                Uwajima, Takayuki                                                             (B) TITLE:Isolation and identification of the Gene                            of Cholesterol Oxidase from                                                   Brevibacterium Sterolicum ATCC 21387 , A                                      Widely used Enzyme in Clinical Analysis                                        (C) JOURNAL:Biochem. Biophys. Res. Commun.                                   (D) VOLUME:172                                                                (E) ISSUE:3                                                                   (E) PAGES:721-727                                                             (G) DATE:30-OCT-1990                                                          (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:Fujishiro, Kinya                                                  Ohta, Toshio                                                                  Hasegawa, Mamoru                                                              Mizukami, Toru                                                                Uwajima, Takayuki                                                             (B) TITLE:Isolation and identification of the Gene                             of Cholesterol Oxidase from                                                  Brevibacterium Sterolicum ATCC 21387 , A                                      Widely used Enzyme in Clinical Analysis                                       (C) JOURNAL:Biochem. Biophys. Res. Commun.                                    (D) VOLUME:172                                                                (E) ISSUE:3                                                                   (E) PAGES:721-727                                                             (G) DATE:30-OCT-1990                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TCRTCDATYTGYTCCAT 17                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:17 base pairs                                                      (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Other nucleic acid Synthetic DNA                           (iii) HYPOTHETICAL:YES                                                        (iv) ANTI-SENSE:YES                                                           (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:Fujishiro, Kinya                                                  Ohta, Toshio                                                                  Hasegawa, Mamoru                                                              Yamaguchi, Kazuo                                                              Mizukami, Toru                                                                Uwajima, Takayuki                                                             (B) TITLE:Isolation and identification of the Gene                            of Cholesterol Oxidase from                                                   Brevibacterium Sterolicum ATCC 21387 , A                                      Widely used Enzyme in Clinical Analysis                                       (C) JOURNAL:Biochem. Biophys. Res. Commun.                                    (D) VOLUME:172                                                                (E) ISSUE:3                                                                   (E) PAGES:721-727                                                             (G) DATE:30-OCT-1990                                                          (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:Fujishiro, Kinya                                                  Ohta, Toshio                                                                  Hasegawa, Mamoru                                                              Mizukami, Toru                                                                Uwajima, Takayuki                                                             (B) TITLE:Isolation and identification of the Gene                             of Cholesterol Oxidase from                                                  Brevibacterium Sterolicum ATCC 21387 , A                                      Widely used Enzyme in Clinical Analysis                                       (C) JOURNAL:Biochem. Biophys. Res. Commun.                                    (D) VOLUME:172                                                                (E) ISSUE:3                                                                   (E) PAGES:721-727                                                             (G) DATE:30-OCT-1990                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATDATYTTRTCCATRTT 17                                                      

What is claimed is:
 1. An isolated and purified cholesterol oxidasewhich has the amino acid sequence as defined in the Sequence Listing bySEG ID NO.: 1, and the following physicochemical properties:(a) activitywhich catalyzes the reaction of oxidizing cholesterol in the presence ofoxygen to form hydrogen peroxide and 4-cholesten-3-one; (b) anisoelectric point at a pH of 4.7; (c) substrate specificity so that theenzymeacts on cholesterol, β-sitosterol, stigmasterol, pregnenolone,dehydroisoandrosterone and estradiol but does not act on vitamin D₃,cholic acid, androsterone, cholesterol linoleate or lanosterol; (d) aworking pH is a range of 5.0 to 7.5, and the enzyme is stable in a pHrange of 5.3 to 7.5 when heated at 50° C. for 60 minutes; (e) optimumtemperature of about 50° C.; (f) the enzyme is inactivated at a pH of10.0 or more or at pH of 4.0 or less then heated at 50° C. for an hourand the enzyme is also inactivated by about 83% when heated at a pH of7.0 and a temperature of 60° C. for an hour; (g) the enzyme is inhibitedby p-chloromercury benzenesulfonate, silver nitrate ando-hydroxyquinoline and in the presence of bovine serum albumin,resistance to heat and stability during storage are improved; (h)Michaelis' constant to cholesterol (Km value) of 3.0×10⁻⁵ M; and (i)molecular weight of about 43,000 (gel filtration), about 60,000(electrophoresis), and 66,586 (DNA sequence).
 2. The isolated andpurified cholesterol oxidase according to claim 1, wherein saidcholesterol oxidase is derived from Brevibacterium sterolicum.